WO1987001708A2 - Bioactivite amelioree de la somatotropine mammifere par desamidation selective - Google Patents

Bioactivite amelioree de la somatotropine mammifere par desamidation selective Download PDF

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WO1987001708A2
WO1987001708A2 PCT/US1986/001860 US8601860W WO8701708A2 WO 1987001708 A2 WO1987001708 A2 WO 1987001708A2 US 8601860 W US8601860 W US 8601860W WO 8701708 A2 WO8701708 A2 WO 8701708A2
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somatotropin
bst
species
bovine
asparagine
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PCT/US1986/001860
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WO1987001708A3 (fr
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Gary C. Harbour
John G. Hoogerheide
Robert L. Garlick
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The Upjohn Company
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Priority to AT86905640T priority Critical patent/ATE57937T1/de
Priority to DE8686905640T priority patent/DE3675382D1/de
Priority claimed from EP19860307767 external-priority patent/EP0263206B1/fr
Publication of WO1987001708A2 publication Critical patent/WO1987001708A2/fr
Publication of WO1987001708A3 publication Critical patent/WO1987001708A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin

Definitions

  • This invention particularly discloses a composition comprised of bovine somatotropin (bSt) compounds wherein the bSt has been modified by conversion from the native state comprising a predominant electrophoretic species having an isoelectric point (pI) of 8.2 to a bioenhanced composition of bSt molecules having a predominant electrophoretic species with a pI between 7.5 and 6.5.
  • the preferred bovine somatotropin is that species modified at the asparagine located between amino acid residues 96-101 to an isoaspartic acid, aspartic acid or glutamic acid (see Figure 1). Chemical and recombinant genetic processes for the production of these bioenhanced compositions are also disclosed.
  • Bovine somatotropin is a growth hormone that has been well studied and the literature has been recently reviewed by Paladini, A.C. et al., Molecular biology of growth hormones. CRC Critical Reviews in Biochem. 15:25-56 (1983). Somatotropins were originally discovered in pituitary gland extracts from various animal species. In general, somatotropins are conserved molecules and similarities in amino acid sequences and structure are found between animals of disparate evolutionary ranking.
  • Growth hormones including bovine growth hormone are globular proteins comprised of a single chain of approximately 200 amino acids, having 2-3 intramolecular disulfide bonds. Accordingly, bSt has a single chain of 190-191 amino acids, globular structure with two intramolecular disulfide bonds and a molecular weight of approximately 22,000 daltons.
  • Natural bSt extracted from pituitary material is a heterogeneous mixture of proteins. At least six major forms of the protein have been described. The longest form is amino-terminal ala-phe bSt having 191 amino acid residues. The second form is amino terminal phe bSt having 190 amino acid residues. The third form is amino terminal Met-bSt having 187 amino acid residues. The remaining three forms of bSt arise where an allelic variation substituting valine for leucine at position 127 in the chain is present.
  • undefined heterogeneity of bovine somatotropin has also been described. Hart, I.C. et al., The heterogeneity of bovine growth hormone. Biochem. J. 218:573-581 (1984); and in Wallace, M. and Dickson, H.B.F. A chromatographic preparation of ox growth hormone. Biochem. J. 100:593-600 (1965).
  • This invention discloses a process for .increasing the bioactivity of bSt through selective deamidation by subjecting the bSt molecules to mild heat and alkaline conditions.
  • the general process involves the conversion of asparagine to aspartic acid or isoaspartic acid in a nonenzymatic acid or base catalyzed reaction. The rate of conversion is dependent in part upon neighboring amino acids in the polypeptide sequence.
  • Bovine somatotropin is known to have sites that are capable of undergoing deamidation. Conditions for in vitro deamidation and the kinetics of deamidation have been described by Lewis, U.J., et al., Kinetic study of the deamidation of growth hormone and prolactin. Biochim. Biophys. Acta, 214:498-508 (1970). It is known for bSt that as pH increases, conversion to lower pl species occurs more rapidly and that as ionic strength is increased in a buffer at pH 10 the rate of deamidation will likewise increase.
  • Bovine somatotropin produced by recombinant microorganisms or extracted from pituitary gland tissue has great commercial value. It increases lactation in dairy cattle and increases size and meat production in beef cattle. Administration to cattle is a problem. It is estimated that upwards to 100 mg per animal per day will be needed to effect commercially acceptable improvements in production. Such a dosage will require efficient methods of administration. Improvements in the potency of bSt such as described in this inven- tion will be of benefit because of resulting reductions in the amount of drug administered to each animal per day.
  • This invention relates to the enhancement of bioactivity for mammalian somatotropins including bSt. It is disclosed herein that the bioactivity of mammalian somatotropins can be enhanced by selective deamidation. More specifically, this invention discloses that milk production and growth rates in animals are enhanced by partially deamidating the bSt molecules prior to administration. The chemical modification described in this invention results in an increase in the proportion of electrophoretic species of bSt having an approximate pi of 7.0. In order to increase the presence of these species, the native forms are incompletely deamidated using mild alkaline conditions and heat.
  • the selectively deamidated species of bSt are capable of increasing growth rate, milk production, and numbers of mammary parenchymal cells in animals at a greater rate than an identical amount of a native pituitary bSt or recombinant bSt.
  • this invention describes a composition comprised of bovine somatotropin-like compounds having a mixture of electrophoretic species wherein the dominant species have isoelectric points of between 7.5 to 6.5.
  • This invention also embraces a partially deamidated species of mammalian somatotropins wherein the asparagine located between amino acid residues 96-101 is converted to isoaspartic acid or aspartic acid.
  • mammalian somatotropins contain an asparagine at the residues 96-101 of their amino acid sequence
  • porcine and human growth hormones in addition to bSt do contain a single asparagine at the appropriate location. Accordingly, those native mammalian somatotropins having an asparagine at this region, which corresponds to a ⁇ turn, are embraced by this invention.
  • analogs of bSt and other mammalian somatotropins having an asparagine in a region corresponding to the 96-101 residues of the native somatotropins are embraced by this invention.
  • Such analogs include somatotropin-like compounds which embrace all proteins having sufficient similarity in amino acid sequence to elicit a growth response in the hypophysectomized rat assay or in the mammal from which it was derived.
  • somatotropins Due to the polymorphism of somatotropins, the position numbers of amino residues of the various somatotropins may differ.
  • the term "native mammalian somatotropin" embraces these naturally occurring species.
  • figure 1 illustrates the specific region of one species of bSt that corresponds to the preferred modification site of this invention. The numbering for other somatotropins may differ where analogs are involved and it is helpful to note that the asparagine of interest appears within a ⁇ turn which is calculated according to Chou and Fasman, J. Mol. Biol. 115:135 (1977) and Ann. Rev. Biochem. 47:251 (1978).
  • a process is also disclosed for enhancing the growth rate of animals by treatment with an effective amount of bovine somatotropin that has been modified to contain a preponderance of electrophoretic species having isoelectric points of between 7.5 to 6.5.
  • this process of increasing the growth rate of animals is more specifically disclosed for use in bovine-type animals.
  • a method of use is also disclosed for increasing the quantity of mammary parenchymal cells in a female ruminant that comprises treatment of the ruminant at a developmental stage of between at about the onset of puberty and the first parturition with an effective amount of bovine somatotropin that has been modified to contain a preponderance of electrophoretic species having isoelectric points of between 7.5 to 6.5.
  • the above method is more particularly described wherein the modified bovine somatotropin is administered between at about the onset of puberty until the first conception.
  • a method of use is also disclosed for increasing milk production in female ruminants comprising the administration of an effective amount of bovine somatotropin that has been modified to contain a preponderance of electrophoretic species having isoelectric points of between 7.5 to 6 . 5. Lastly this method is described more particularly wherein the ruminant is a dairy cow.
  • deamidated or deamidation refer to chemically or recombinant genetically introduced modifications to a somatotropin which result in the conversion of an asparagine to an isoaspartic acid, aspartic acid, or glutamic acid. Chemically introduced modifications result in isoaspartic acid or aspartic acid in place of asparagine. Recombinant genetically introduced modifications result in either an aspartic acid or glutamic acid residue in place of asparagine.
  • the disclosed chemical treatments initially deamidate the preferred asparagine residue but will with time deamidate any available asparagine or glutamine.
  • the conversion of an asparagine residue to a negatively charged residue will alter the somatotropin's profile on an isoelectric focusing gel and can be monitored using isoelectric focusing methods.
  • partial or selective deamidation refers to incompletely deamidated somatotropin-like proteins having an isoelectric point between 7.5 and 6.5.
  • closest-related native somatotropin refers to the naturally-occurring form of mammalian somatotropin which when compared to a specific somatatropin-like protein is more closely identical in amino acid sequence than any other naturally-occurring form of mammalian somatotropin.
  • somatotropin-like protein refers to both native forms of somatotropins and to analogs of native somatotropins provided that the analogs have sufficient protein identity with their parent compounds to demonstrate bioactivity as either a growth promoter or as a stimulant for milk production.
  • mammalian somatotropin refers to somatotropins originating from mammals and includes somatotropins derived from either natural sources, e.g., pituitary gland tissue or from microorganisms transformed by recombinant genetics to produce a naturallyoccurring form of somatotropin.
  • a specific mammalian source such as a bovine somatotropin or a somatotropin of bovine origin
  • the somatotropin includes those derived from either natural sources or from transformed microorganisms.
  • microorganism refers to both single cellular prokaryote and eukaryote organisms such as bacteria, yeast, actinomycetes and single cells from higher order plants or animals when being grown in cell culture.
  • somatotropins refers to naturally-occurring forms of somatotropins which may have been derived from either natural sources, e.g., pituitary gland tissue or from microorganisms transformed by recombinant genetics to produce a naturally-occurring form of somatotropin.
  • vector includes both cloning plasmids and plasmids which are capable of directing the expression of a somatotropin by virtue of the cDNA encoding the somatotropin being operatively linked to a promoter capable of being recognized by an microorganism.
  • the mammalian somatotropins are very similar in amino acid sequence and physical structure. Although the processes described below are directed towards the deamidation of bSt, the processes are equally applicable to any mammalian somatotropin having the requisite asparagine residue available for deamidation.
  • the ionic species in a typical sample of bSt from a pituitary gland or recombinant microorganism range from isoelectric points of 9.0-5.5.
  • the chemically modified bSt is also a mixture of species; however, the species having isoelectric points inside the bioenhanced region of 7.5 to 6.5 predominate.
  • the basis of this invention resides in the determination that the natural electrophoretic heterogeneity of bSt can be altered by deamidation to yield an increased proportion of species having a pi of approximately 7.0 to achieve an increased biological effect. Hart, et al. and Wallace and Dickson, supra.
  • bSt for chemical deamidation is a species produced by a recombinant microorganism because it is less heterogeneous than pituitary extracted bSt and more economical to produce.
  • the most preferred deamidated species of bSt having an approximate pi of 7.0 has been selectively modified at the asparagine located between residues 96-101 to isoaspartic acid.
  • Analogs of bSt are known (see European patent applications 75,444 and 103,395). Such analogs are also capable of being modified to enhance bioactivity and are considered a part of this invention.
  • the processes for chemically modifying somatotropin-like proteins include acid treatment or base treatment.
  • the treatment described below partially deamidates bSt and is preferred. Efficient deamidation is achieved by alkaline treatment and elevated temperatures. A pH range of 7.5 to 11 and a temperature range of 20° to 50oC is generally effective. Ionic strength is also a factor and a range of 0.01 to 2.0 dependent on the particular buffer and salts being present is effective. Without control of the process, as described below, the predominant bSt species will have a pi of 6.0 or below.
  • the duration of chemical treatment varies according to temperature and pH. At elevated temperatures and at the pH extremes, deamidation occurs more rapidly than at low temperatures and neutral pH. Isoelectric focusing or reversed phase high performance liquid chromatography procedures can be used to monitor the process.
  • bSt The most acidic species of bSt (pi 5.0) is the least active species in relative potency. The most potent species are only partially deamidated and fall within the 7.5-6.5 region.
  • mild deamidating methods must be employed. For example, recombinant bSt can be suspended in alkaline buffers such as Tris-HCl, sodium borate, sodium carbonate or sodium phosphate at an ionic strength of 0.01 to 0.2, and heated to 30o-50°C noting that rate of deamidation will increase rapidly with increased temperature. These mild conditions permit partial deamidation to occur slowly over a 4-5 day period.
  • the concentration of recombinant bSt ranges from 1.0 to 10 mg/ml.
  • chromatographic or electrophoretic techniques are optionally used to purify the mixture of ionic species.
  • the aim of the purification is to increase the proportion of ionic species of bSt having an isoelectric point around 7.0 and increase the bioactivity of the final product.
  • Techniques for isolation and purification include preparative isoelectric focusing and ion exchange chromatography with anion exchange chromatography being a preferred technique.
  • the most preferred method of separation is reversed phase high performance liquid chromatography.
  • the preferred species of deamidated bSt has a different retention time from the native species allowing for easy separation and purification.
  • the high relative potency of the deamidated bSt is readily determined using hypophysectomized rats. Evans, H.M. and Long J.A., The effect of the anterior lobe of the hypophysis administered intraperitoneally upon growth, and the maturity and oestrus cycles of the rat, Anat. Rec. 21:61, 1921. Relative increases in total body weight are recorded using pituitary bSt, recombinant bSt [rbSt] and various fractions of deamidated rbSt. Results in rats using ionic species of deamidated rbSt having a pi of approximately 7.0 demonstrate a 2-3 times greater potency than native undeamidated bSt.
  • Administration into dairy cattle is according to known methods using any route effective to deliver the required dosage to the animal's circulatory system.
  • Modes of administration include oral, intramuscular injections, subcutaneous injections and the use of timed-release implants.
  • the preferred mode of administration is by subcutaneous injection using a timed-release implant.
  • Appropriate vehicles for injections include physiologically compatible buffers such as sodium bicarbonate, sodium phosphate, or ammonium phosphate solutions.
  • Timed-release implants are known in the art, e.g., U. S. Pat. 4,333,919.
  • the effective dosage range is from 1.0 to 200 milligrams per animal per day.
  • Mammalian growth hormones are very similar in their amino acid sequences and hormones originating from one animal source can enhance the growth of other unrelated species of animals.
  • deamidated bSt can be used to produce increased growth in the same animal species in which native bSt has been shown to have growth-related bioactivity such as bovines, sheep, rats, salmon and chickens.
  • the preferred animals are bovine used for beef cattle such as bulls, heifers or steers.
  • Deamidated bSt can be used to produce increased growth rates in beef cattle by administration any time between weaning until slaughter. Deamidated bSt is administered to beef cattle for a minimum of 30 days and for a maximum of 450 days depending upon desired time of slaughter. Animals used for veal are typically slaughtered at approximately 6 months of age and 10 to 30 mg/day of deamidated bSt is administered up until the age of slaughter to effectuate desired increases in growth rate.
  • deamidated bSt is administered between 30 and 90 days post-partum and continued for up to 300 days. Deamidated bSt will also increase lactation in other commercial milk-producing animals such as goats or sheep.
  • the methods described in U.S. Patent 4,521,409 are incorporated by reference herein. Briefly the ruminants which include dairy heifers, sheep and goats among others are treated with the deamidated bSt sometime around the onset of puberty. Treatment is continued until first conception or parturition. More particularly administration of deamidated bSt in dairy heifers can be begun within 30 days of the expected onset of puberty and continued up until 100 days after the onset of puberty.
  • Puberty is not determinable by visual inspection. Puberty is defined by the period beginning with the initiation of the sexual process and lasting up until the maturation of the sexual organs. The precise time of administration will depend upon the particular breed of animal, its nutritional status and management practices.
  • Recombinant bSt is known and can be produced by a number of published procedures such as described in Schoner, B.E., et al., Role of messenger RNA translational efficiency in bovine growth hormone expression in Escherichia coli. PNAS, USA, 81:5403-5407 (1984); Seeburg, P.H. Efficient bacterial expression of bovine and porcine growth hormones. DNA, 2:37-45 (1983) and European patent application 75,444.
  • the choice of rbSt species is not relevant to this invention.
  • N-terminal heterogeneiety is known to exist for bSt. Two species exemplified herein are the amino-terminal ala-phe- and phe- forms of rbSt.
  • An alternative process for partially deamidating bSt uses a buffer of 100 mM sodium phosphate at pH 9.45 having a concentration of bSt of 1-2 mg/ml which is then heated at 37o for 96 hours . This material is then dialyzed against dilute NaOH, followed by lyophilization.
  • the concentration of acetonitrile is 35%.
  • the concentration of solvent B increases to 50% over 30 minutes and then is maintained at 50% for another 15 minutes, after which the B solvent is increased to 100% over 5 minutes.
  • the rbSt component which is modified at asparagine residue 99, elutes approximately 2 minutes earlier than native rbSt.
  • the relative retention time of the modified rbSt is approximately 0.94, when compared to the retention time of native rbSt.
  • reversed phase HPLC is conducted using two 0.41 x 25 cm Vydac C-4 columns connected in series, and the same solvent gradient as is used in analytical studies.
  • up to 10 mg rbSt is applied to the columns for each run.
  • the relative retention time for the modified rbSt component, compared to native rbSt is approximately 0.90.
  • the fractions containing deamidated recombinant bSt having a pi of 7.5 to 6.5 are then collected, made 6 M in urea, titrated to pH 10-10.5 with ammonium hydroxide, dialyzed against dilute NaOH and freeze dried.
  • Changes in the DNA sequence of rbSt are made by site-directed mutagenesis using the methods of Zoller and Smith (Nucl. Acid Res. 10:6487-6500,1982 and Methods in Enzymology 100:468-500, 1983).
  • a segment of DNA from the rbSt gene is cloned into M13mp phage vectors and used to infect E. coli.
  • Single stranded DNA can be isolated from the M13 phage virions, and hybridized with a synthetic oligomer which contains the desired base changes.
  • a double stranded region is isolated after primer extension by digestion with restriction endonucleases and cloned into a "transition vector".
  • Example 3A is based on the modified rbSt gene in the expression vector pBGH33-4, as described in the Genentech European Patent application 75444 and Great Britain Pat. No. 2.147.902B. This gene has been modified in its N-terminal DNA sequence to enhance expression of the rbSt protein. This example should not be construed as a limitation upon the disclosed invention.
  • the use of pBGH33-4 to exemplify site specific mutations of rbSt at codon 98 is a matter of convenience.
  • pBGH33-4 contains the phe-rbSt gene and amino acid references to position 98 correspond to position 99 in the ala-phe-rbSt used for chemical deamidation and biological testing.
  • Example 3B discloses a convenient method for inserting an alanine into the codon at position 1 of phe-rbSt cDNA.
  • Example 3A The introduction of glutamic or aspartic acid at position "99" of phe-rbSt.
  • the purpose of the transition vector is to provide a vector which has manipulatable restriction sites for reconstruction of the intact rbSt gene after a sub-segment has been mutagenized.
  • this example takes advantage of a PstI restriction site in the rbSt cDNA sequence located upstream of the 98th codon. This PstI site is unique in the gene but is not unique for the Genentech vector pBGH33-4.
  • a Pstl/BamHI restriction fragment encompassing this sequence is cloned into the M13mpl9 vector (Messing, Methods in Enzymology 101:20-78,1983 and Yanisck-Perron et al., Gene 33:103-119, 1985). These M13mp vectors are commercially available (International Biotechnology, Inc., New Haven, Connecticut, USA). After mutagenesis of the Pstl/BamHI rbSt sub-segment, the rbSt gene is cloned into a transition vector in which the PstI and BamHI restriction sites of the original rbSt gene are unique. The construction of pUC19(d) - Chart 1.
  • the pUC19 vector (International Biotechnology, Inc.), contains a single PstI site located in a polylinker (Yanisch-Perron et al., 1985 supra). The site lies between the Hinll and Hindlll restriction sites.
  • the PstI site is deleted by cutting purified DNA at the Hinll and Hindlll sites, filling the 5' overhang of the Hindlll site with Poll Klenow (Pollk), purifying the large cut vector fragment by agarose gel electrophoresis and electroelution, ligating with T4 DNA iigase and ATP, and transforming competent cells selecting for resistance to ampicillin.
  • Verification of the presence of pUC19(d) in a clone is obtained by isolating DNA from several clones using the method of Birnboim and Doly (Nucl. Acid Res. 7:1513-1523,1979). The isolated DNA is then analyzed by restriction digestions for vectors which are not cut by Hindll, Hinll, or PstI and are cut once by Xbal and BamHI. These procedures are described in detail in Maniatis et al., Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory, 1982. The resultant vector is designated as pUC19(d).
  • the entire rbSt gene is isolated from the vector pBGH33-4 on an Xbal, BamHI restriction fragment 1 (0.9 kb) and cloned into the complementary Xbal, BamHI restriction sites of pUC19(d).
  • fragment 1 (0.9 kb) is isolated from pBGH33-4
  • fragment 2 (2.7 kb) is isolated from pUC19(d) after the individual vectors are digested with Xbal and BamHI.
  • the fragments are cut from a gel and isolated by electroelution. The steps involved are the same as those described for the construction of pUC19(d).
  • the resultant vector is designated as pUC19(d)-bSt.
  • the large fragment (about 7.2 kb) is purified and ligated to fragment 3 to yield pM13-bSt'.
  • E. coli cells are transfected and individual plaques are isolated.
  • the RF DNA is isolated again and the insertion of the fragment is confirmed by analytical digestions with restriction endonucleases.
  • the PstI restriction site for rbSt cDNA corresponds to amino acids 89 and 90 of rbSt.
  • the target codon, 98 is located within the PstI, BamHI fragment.
  • B. Modification of position 98 Design of DNA Oligomers Two synthetic oligonucleotides, 39 nucleotides in length are shown in figure 2.
  • Hybridization conditions are described by Zoller and Smith (Methods in Enzymology 100:468-500, 1983).
  • An oligomer with the desired codon change at position 98 is hybridized in a 20 to 30 molar excess to its complement with 1 pmole of the single stranded DNA molecules by heating at 65°C and allowing the mixture to cool to room temperature. Primer Extension.
  • the modified rbSt gene can now be recloned as an Xbal, BamHI fragment from the transition vector, pUC19(d) -bSt*, into the Genentech expression vector pBGH33-4.
  • the transition vector is digested with the restriction enzymes Xbal and BamHI and fragment 7 (1.0 kb) is obtained. Fragment 7, which contains the entire coding sequence for rbSt, is isolated.
  • the rbSt expression vector pBGH33-4 is digested with the same enzymes and the vector fragment 8 (4.0 kb) is isolated. The two fragments are ligated and transformed into competent cells.
  • the plasmid pBGH33-4 contains an Xbal restriction site located between the ribosomal binding site and the ATG initiation codon of the rbSt gene. Approximately 72 nucleotides downstream of this site there is a PvuII restriction site. A synthetic double stranded oligomer can be inserted between these two restriction sites which contains the additional ala codon at the second amino acid position.
  • the PvuII restriction site is not unique for the gene. This problem can be circumvented by constructing a vector containing only that portion of the rbSt gene which is to be modified and in which this PvuII site is unique. The front end of the gene is modified and an intact rbSt gene Is reconstructed. The construction of pUC8-bSt'- Chart 6.
  • Plasmid pUC8-bSt' is constructed by digesting the DNA of pBGH334 with EcoRI and PstI restriction endonucleases. Fragment 9 (550 bp) is cut from an agarose gel and electro-eluted. This fragment contains the trp promoter, a ribosomal binding site and the front half of the rbSt gene.
  • the pUC8 vector (commercially available from Bethesda Research Laboratories, Gaithersburg, Maryland, USA) is digested with EcoRI and PstI and the 2.7 kb vector fragment 10 is isolated. The two purified DNA molecules are ligated and transformed into competent E. coli cells. Cells containing the vectors are selected by resistance to ampicillin.
  • This resultant vector is designated pUC19(d)-bSt-ala and can be used as the transition vector for the position 98 base changes following the steps described in Example 3A above.
  • Example 4. Biological Assay. Samples of deamidated rbSt are treated for relative potency using the hypophysectomized (pituitary surgically removed) rat bioassay. Two hundred hypophysectomized female Sprague Dawley rats weighing between 75 and 150 grams are used in each experiment. They are fed ad libitum pelleted Purina Rat Chow which is a commercial product and watered with deionized and rechlorinated water. Room conditions are set at a temperature of 80oF and relative humidity of 50%.
  • Stock solutions of rbSt at 2 mg/ml are prepared in a buffer of 0.03M NaHCO 3 and 0.15M NaCl at pH 9.5. To facilitate suspension of rbSt, the lyophilized preparations are first dissolved in this buffer at pH 10.8, pH is then adjusted to 9.5 using 2N HCl and brought to final volume using the stock buffer at pH 9.5 and filtered if necessary.
  • Example 6 To increase the milk production in dairy cows, cows sixty days post-partum are selected for treatment. Each animal receives recombinant deamidated bovine somatotropin administered subcutaneously at a rate of 50 milligrams per day for 300 days. The animals are permitted to eat and drink at will.
  • FIGURE 1 The following nucleotide and amino acid sequence is from
  • European Patent Application 75,444 It represents a hybrid bSt molecule and is useful herein to point out the relevant asparagine within the specific ⁇ turn region of interest. (See underlined residue 981)
  • Plasmid pBGH 33-4 is cut with PstI and BamHI and fragment 3 (700 kb) containing the 3' end of the bSt gene is isolated.
  • Fragments 5 and 6 are ligated and the resulting heteroduplex is transformed into E. coli which forms two populations of homoduplexes.
  • the homoduplex containing the appropriate modification at position 98 (aspartic acid) is selected by gel sequencing and isolated for expression.
  • the vector is designated as pUC19(d)-bSt*.
  • Fragments 7 and 8 are ligated. to form the expression vector pBGH33-4-"Glu98" (5.8 kb).
  • Fragment 12 (72 bp), a double stranded oligonucleotide containing the 5' portion of bSt and incorporating an alanine codon at position one, is artificially synthesized.
  • Fragments 11 and 12 are ligated with T4 ligase to yield pUC8-bSt'ala.
  • B Bovine somatotropin cDNA.
  • A Alanine codon.
  • B Bovine somatotropin cDNA.
  • A Alanine codon.
  • This invention parti ularly discloses a composition comprised of bovine somatotropin (bSt) compounds wherein the bSt has been modified deamidation from the native state comprising a predominant electrophoretic species having an isoelectric point (pi) of 8 to a bioenhanced composition of bSt molecules having a predominant electrophoretic species with pi between 7.5 and 6.
  • the preferred bovine somatotropin is that species in which the asparagine located between amino acid residues 96-101 h been modified to isoaspartic acid. Processes for the production of these bioenhanced compositions and for their use are so disclosed.

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Abstract

Des formes désamidées de somatotropines mammifères ci-décrites ont une asparagine située entre les résidus (96-101). Ces somatotropines désamidées ont une bioactivité accrue par rapport à leurs espèces natives. Cette invention décrit en particulier une composition consistant en des composés de somatotropine bovine (bSt) où bSt a été modifié par désamidation d'un état natif comprenant une espèce électrophorétique prédominante ayant un point isoélectrique (pI) de 8,2 en une composition bioaméliorée de molécules bSt ayant une espèce électrophorétique prédominante avec un pI compris entre 7,5 et 6,5. La somatotropine bovine préférée est une espèce dans laquelle l'asparagine située entre les résidus d'acide aminé (96-101) a été modifiée en acide isoaspartique. Des procédés de production de ces compositions bioaméliorées et leur utilisation sont également décrits.
PCT/US1986/001860 1985-09-18 1986-09-15 Bioactivite amelioree de la somatotropine mammifere par desamidation selective WO1987001708A2 (fr)

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AT86905640T ATE57937T1 (de) 1985-09-18 1986-09-15 Erhoehte biowirkung von saeugetier-somatotropin durch selektive deamidierung.
DE8686905640T DE3675382D1 (de) 1985-09-18 1986-09-15 Erhoehte biowirkung von saeugetier-somatotropin durch selektive deamidierung.

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US77715485A 1985-09-18 1985-09-18
US777,154 1985-09-18
EP19860307767 EP0263206B1 (fr) 1986-10-08 1986-10-08 Bioactivité élevée de la somatotropine des mammifères par désamidation sélective

Publications (2)

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WO1987001708A2 true WO1987001708A2 (fr) 1987-03-26
WO1987001708A3 WO1987001708A3 (fr) 1987-08-13

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PCT/US1986/001860 WO1987001708A2 (fr) 1985-09-18 1986-09-15 Bioactivite amelioree de la somatotropine mammifere par desamidation selective

Country Status (5)

Country Link
EP (1) EP0236452B1 (fr)
JP (1) JPS63500941A (fr)
AT (1) ATE57937T1 (fr)
DE (1) DE3675382D1 (fr)
WO (1) WO1987001708A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0333438A2 (fr) * 1988-03-15 1989-09-20 Eli Lilly And Company Dérivé de l'hormone de croissance humaine
WO1990002182A1 (fr) * 1988-08-29 1990-03-08 Monsanto Company Nouvelles somatotropines
WO1990008164A1 (fr) * 1989-01-19 1990-07-26 The Upjohn Company Analogues de somatotrophine
US5631227A (en) * 1985-09-18 1997-05-20 The Upjohn Company Somatotropin analogs

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0047600A2 (fr) * 1980-08-26 1982-03-17 The Regents Of The University Of California Prohormone et hormone de croissance bovine
EP0131843A1 (fr) * 1983-07-15 1985-01-23 Bio-Technology General Corporation Vecteurs d'expression pour la production accrue de polypeptides, plasmides contenant ces vecteurs, hôtes contenant ces plasmides, produits manufacturés par ces vecteurs et méthodes correspondantes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0047600A2 (fr) * 1980-08-26 1982-03-17 The Regents Of The University Of California Prohormone et hormone de croissance bovine
EP0131843A1 (fr) * 1983-07-15 1985-01-23 Bio-Technology General Corporation Vecteurs d'expression pour la production accrue de polypeptides, plasmides contenant ces vecteurs, hôtes contenant ces plasmides, produits manufacturés par ces vecteurs et méthodes correspondantes

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5631227A (en) * 1985-09-18 1997-05-20 The Upjohn Company Somatotropin analogs
EP0333438A2 (fr) * 1988-03-15 1989-09-20 Eli Lilly And Company Dérivé de l'hormone de croissance humaine
EP0333438A3 (en) * 1988-03-15 1989-11-29 Eli Lilly And Company Novel derivative of human growth hormone
WO1990002182A1 (fr) * 1988-08-29 1990-03-08 Monsanto Company Nouvelles somatotropines
US5089473A (en) * 1988-08-29 1992-02-18 Monsanto Company Somatotropin variants and their use
WO1990008164A1 (fr) * 1989-01-19 1990-07-26 The Upjohn Company Analogues de somatotrophine
AU634517B2 (en) * 1989-01-19 1993-02-25 Pharmacia & Upjohn Company Somatotropin analogs

Also Published As

Publication number Publication date
EP0236452B1 (fr) 1990-10-31
JPS63500941A (ja) 1988-04-07
WO1987001708A3 (fr) 1987-08-13
ATE57937T1 (de) 1990-11-15
EP0236452A1 (fr) 1987-09-16
DE3675382D1 (de) 1990-12-06

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